Project Summary Endothelial barrier dysfunction is a central factor in the pathogenesis of Acute Respiratory Distress Syndrome (ARDS) and Acute Lung Injury (ALI). In recent years, considerable advances have been made in the understanding of how intracellular signaling pathways modulate the disruption and assembly of adherens junctions (AJs). Re-annealing of AJs is a metabolically active process. Yet, little is known about the role of endothelial metabolism as a modulator of endothelial barrier function and restoration of lung vascular injury. Our Supporting Data demonstrate that endothelial cells respond to inflammatory activation with upregulation of signaling via the hypoxia-inducible factor HIF1?, and its crucial downstream metabolic target PFK-FB3, a critical regulatory enzyme for glycolysis. This glycolytic shift is accompanied by concomitant upregulation of mitochondrial glutamine metabolism, which compensates for the loss of mitochondrial glucose oxidation and enables cells to use glutamine as an alternate mitochondrial TCA cycle fuel. We observed that inhibition of PFK- FB3 prevents restoration of endothelial barrier function following lung injury, thus underscoring the adaptive role of PFK-FB3 and increased glycolysis during endothelial barrier restoration. Based on these findings, we posit that induction of glycolysis in lung microvessel endothelial cells serves as a homeostatic mechanism mediating the restoration of endothelial barrier function and lung fluid balance. In Project 2, we will pursue the following Specific Aims: (1a) We will define the mechanisms of PFK-FB3-mediated activation of glycolysis and compensatory glutaminolysis in lung endothelial cells as induced by inflammation and endothelial injury, and determine the requisite role of these metabolic shifts in repairing endothelial barrier; (1b) we will determine the spatial-temporal role of PFK-FB3-mediated activation of glycolysis in the re-annealing of AJs and restoring endothelial barrier integrity, and (2) We will determine the role of endothelial metabolic reprogramming via PFK-FB3 in restoring lung endothelial barrier integrity and fluid balance following inflammatory lung injury in models of ALI. Using state-of-the-art metabolic analyses, engineered protein constructs and biosensors as well as novel genetic mouse models, we will define the metabolic mechanisms activated by inflammatory injury of the lung endothelium and their role in restoring the lung endothelial barrier. Our long-term goal is to identify metabolic targets and switches that will promote and accelerate the recovery of the endothelial barrier and normalize lung fluid balance to mitigate acute lung injury.